drazones tested exhibited a fluorescence peak a t 470 mp when excited at 390 to 400 n i ~ . p-Aminoacetophenone, while fluorescing a t this wave length, ivas excited maximally by a shorter excitation wive length. Fluorescence spectra of these four compounds were easily obtained even with an excess of salicyloyl hydrazide. A reaction mixture of one of the above-mentioned substances and salicyloyl hydrazide could be read directly in the spectrophotofluorometer, after condmsation in acetic acid-ethyl alco-
hol with heating. The mixture ncetltd only to be neutralized, brought to an alkaline pH with buffer, diluted properly, and read in the instrument. Fluorescent salicyloyl hydrazones which cannot be resolved by their fluorescence spectra could be assayed, if free salicyloyl hydrazide was removed after reaction. One simple method is to partition between purified dichloromethane (purified through silica gel) and 20% ethyl alcohol, 0.1M in hydrochloric acid. Salicyloyl hydrazide remains in the aqueous phase.
LITERATURE CITED
( I ) Bowman, R. L., Caulfield, P. A , , Cdenfriend, Sidney, Science 122, 32-3 (1955). (2]-Camber, B., Nature 174, 1107 (1954). ( 3 ) Duggan, D. E., Bowman, R. L.,
Bradie, B. B., Udenfriend, Sidnev, Arch. ' Biochem. B i o p h y s . 68, 1-i4
(1957). ( 4 ) Udenfriend, Sidney, Duggan, D. E., Vasta, B. II.,Brodie, B. B., J . Pharmacol. Ezptl. Therap. 120, 26-32 (1957).
RECEIVED for revie\? January 18, 1958. Accepted October 14,1958.
Enzymatic Determination of Polyunsaturated Fatty Acids JOSEPH MacGEE Miami Valley laboratories, The Procter & Gamble Co., Cincinnati, Ohio
b A simple and rapid enzymatic method for the quantitative estimation of total cis-methylene-interrupted polyenoic acids has been devised. Linoleic, linolenic, and arachidonic acids, the more common acids of this group, were used to calibrate the method. The potassium salts of the fatty acids are oxidized b y atmospheric oxygen in the presence of the enzyme lipoxidase, and the absorption of the conjugated diene hydroperoxide is measured a t 234 mp. As little as 5 y of linoleic acid can b e quantitatively measured with good accuracy and precision. The total content of polyunsaturated fatty acids containing the cis-methylene-interrupted diene structure of fats, oils, hydrogenated oils, fatty acids, esters, blood plasma, microorganisms, and plant seeds has been measured directly by this method.
T
HL quantitative estimation of polyuiisaturated fatty acids in the fats and oils of commerce and in tissues has, a t best, been difficult. Before publication of the ultraviolets pectrophotometric method developed by NIitchell, Kraybill, and Zscheile (6), the principal method for the determination of these biologically important acids involved physical isolation of the individual fatty acids by means of their bromide derivatives ( 2 ) . As more information about their functions became available, the importance of polyunsaturated fatty acids in the fields of nutrition and medicine becanie more evident, and methods for their estimation were sought. The first big advance in this analytical problem was the development of the spectrophotometric method based on the absorption of ultraviolet light by tile conjugated unsaturation induced by
298
ANALYTICAL CHEMISTRY
alkaline isomerization of the polyunsaturated fatty acids (6). However, in addition to being strictly empirical, satisfactory application of the isomerization method has been limited to samples which are available in relatively large amounts (about 0.1 gram) as isolated lipides, and to materials with a limited trans-isomer content. The latter point is important, because knowledge of the cis-polyunsaturated fatty acid content is often desired. Although the alkali isomerization technique is quantitative for the cis-isomers, transisomers are partially measured. The need for a simple method which would be specific for cis-isomers and at the same time be applicable to smaller quantities of materials and to the analysis of body fluids and tissues for their polyunsaturated fatty acid content led to consideration of the specific reagent, lipoxidase. Lipoxidase is an oxidative enzyme found in a variety of plant tissues. 18
I7
I6
$5
13
14
I2
/I
10
9
8
Its only known substrates all contain single methylene-interrupted double bonds in which the double bonds are in the cis configuration. Three of the many natural substrates of lipoxidase are the polyunsaturated fatty acids, linoleic, linolenic, and arachidonic. The mode of action of lipoxidase on these acids involves a shift of one of the double bonds and addition of oxygen to form a conjugated diene hydroperoxide (5). Figure 1 illustrates the products from linoleic acid. The activity of the enzyme has been measured, by others, by means of the rates of oxygen uptake, hydroperoxide formation, and conjugated diene formation in the presence of excess substrate, but with limiting amounts of enzyme (4). A method based on ultraviolet absorption after complete conjugation of a limited amount of a substrate in the presence of an excess of enzyme is a logical extension. The alkali isomerization procedure
e
7
I
, ,
2
C H ~ C H ~ C H ~ C H ~ C H ~ CC H = ~CH H = CcHH ~ C H ~ C H ~ C H ~ C H ~ C H ~ C H ~ ~ O O H
Figure 1. Enzymatic oxidation and conjugation of linoleic acid
4 I.
I3
I*
,,
10
I
I
- CHzCH=CHCH=CHyHCH2 OOH
AIO' 12
,
9 - hydroperoxide
w
I,
E
+H10
0
I
.CH2$HCH = C H CH = C HCH2 ,I
OOH
A9,II , 13 -hydroperoxide
measures the percentage of each individual unsaturated fatty acid, while the lipoxidase method measures the total of the polyunsaturated fatty acids, and is thus an adjunct to the available analytical techniques. REAGENTS POTASSIUM BORATEBUFFER. 1.OM, pH 9.0. Dissolve 61.9 grams of boric acid and 25.0 grams of potassium liydroxide in about 800 ml. of distilled water by stirring while heating on a steam bath. After complete solution, allow the solution to cool to room temperature with stirring. Adjust the p H of the solution to 9.0 by addition of 1N potassium hydroxide or 1N hydrochloric acid as required. Adjust the volume of the solution to 1 liter bv adding distilled water. 0.2M. DH 9.0. Transfer 200 ml. of the lk? buffer to a 1-liter volumetric flask, and dilute to volume with distilled water. LIPOXIIJASE. Stock Solution. Dissolve 10 mg. of lipoxidase (Nutritional Biochemical Corp. or General Biochemicals Inc.) in 10 ml. of ice-cold 0.2M buffer. Dilute Solution. Mix 2 ml. of the stock solution with 8 ml. or ice-cold 0.2M buffer. Boiled Dilute Solution. Transfer 5 nil. of the dilute solution to a test tube and hold in a boiling water bath for 5 minutes.
Because the activity of the enzvine may vary from batch to batch, each bottle of enzyme must be tested before use. Prepare a sample solution from cottonseed oil as described under fatty acid esters. Place 3.0-ml. aliquots of this sample solution in each of two 1-cm. quartz cuvettes for the Beckman DE spectrophotometer. To the first cuvette, add 0.10 ml. of the boiled dilute lipoxidase and mix. Use this cuvette to zero the instrument a t 234 mp. Add 0.10 ml. of the dilute lipoxidase (unboiled) to the second curette, mix, and note the time. Place the second cuvette in the instrument and take readings of the absorbance of the second cuvette against the first cuvette as a blank a t 1-minute intervals. The absorbance should increase to a maximum level in the second cuvette in less than 5 minutes. If not, either more concentrated preparations of dilute solution and boilrd dilute solution must be prepared and tested to yield the necessary rate, or a more active supply of lipoxidase is necessary. The enzyme and solutions must bc stored in a freezer. The solutions can be thawed by placing them in a water bath a t room temperature, but they should be held in an ice and water bath as soon as they are completely thawed. The stock solution, dilute solution, and boiled dilute solution have been used after storage for several months n-ithout noticeable deterioration.
PROCEDURE
SAMPLESOLUTION. Prepare a solution in the 0.2M buffer such that each 3.0 ml. contains between 5 and 257 of the free polyunsaturated fatty acids. I n each of two test tubes, place 3.0 ml. of the sample solution. To the first tube (the blank) add 0.10 ml. of the boiled dilute enzyme solution and mix. To the second tube (the sample) add 0.10 ml. of the unboiled dilute enzyme solution and mix. Allow the contents of the tubes t o stand at room temperature for 30 minutes, then transfer to 1cm. quartz cuvettes and place in the Beckman DU spectrophotometer. Set the instrument a t zero with the blank sample and measure the absorbance of the reacted sample solution at 234 mp. Calculate the polyunsaturated fattv acid content from the follon-ing formula : mc PF.\ =
n-here .4
=
TI’
=
SOURCE
x
OF
.4 X 3964 TB
difference in absorbance between the blank and sample total micrograms of sample in 3.0 ml. of sample solution 3964 FACTOR.
=
3.1 3.0 - X
100 x 3
where _ 3‘1 - dilution factor, 3 ml. of 3.0 sample plus 0.1 m ~ of . enzyme 78.2 = specific extinction coefficient (absorbance of 1 gram of PFA per liter in 1-cm. light path at 234 nip) 1000 = conversion from grams per liter to y per ml. 100 = conversion to percentage 3 = volumeof sample APPLICATIONS
Free Fatty Acids. The polyunsaturated fatty acid content of free fatty acids is determined after solution of the acids in 0.2M buffer. This is most conveniently done by adding one fifth of the final volume of the 1.OM buffer to the sample, mixing well, and diluting with distilled water to rolume-yielding a solution in 0.2.M buffer. Fatty Acid Esters. Esters must first be saponified. Saponify a sample containing 0.5 mg. of polyunsaturated fatty acids by mixing it in a 100-ml. volumetric flask 11-ith 1 nil. of 0.55 alcoholic potassium hydroxide, and holding in the dark for at least 4 hours. After saponification, add 20 nil. of the 1 . O N buffer, 1 ml. of 0.5S hydrochloric acid, and water to the 100-nil. mark. DISCUSSION
Several important precautions must be observed to ensure satisfactorv results with the enzymatic analysis of
polyunsaturated fatt acids are highl!- reacti lations of the sample must be rarrietl out in vessels which are flushed with nitrogen-particularly any saponificetions. However, because oxygen is a reactant in the lipoxidase-catalyzwU oxidation and isomerization of polyunsaturated fatty acids, the enzymatic reaction itself must be carried out in an atmosphere of air. If several samples are to he read, care must be taken that the cuvette used for the blank sample does not become contaminated with active enzyme or with any of the test samples containing the active enzyme. Temperature, hydrogen ion concentration, and the presence of organic solvents can profoundly affect enzyme action. It is therefore necessary to neutralize any alkali used in saponification procedures before adding the enzyme to the sample. Any organic solvents used to transfer samples must be removed with the aid of gentle heat and a stream of nitrogen. Up to 3% ethyl alcohol does not affect the procedure described. If absolutely necessary, ethyl alcohol up t o 5% final concentration can be tolerated, but because it is about 50% inhibitory at that concentration, the incubation period with the enzyme must be increased a t least twofold. TT’hen relatively high concentrations of saturated esters or fatty acids are present in the samples, the saponification mixture must be heated briefly to allow good contact between the saponification reagent and the sample or to aid in the solution of the arids in the buffer. Adherence to Beer’s Law. To test the adheience of the lipoxidasc rncthod to Becr’s law, a series of ronrcntiations of saponified cottonseed oil (polyunsatuiated fatty acid content about 50%) m-as subjectcd to thr cnzymatic oxidative procedurc.. The calibration curve showed that Brcr’s law is obeyed. Effect of Temperature on Extinction Coefficient. Holman ( 3 ) states that the products of lipouidase-catalyzed oxidation of polyunsaturated fatty acids vary nith the temperature at 11hich the reaction takes place, because side reactions at the higher temperatures cause the extinction coefficient t o be low. He found the molar extinction coefficient to vary between 31,400 and 23,000, drpending on temperature. To test these findings under the conditions selected for the enzymatic analysis for polyunsaturated fatty acids, samples of linoleic acid were subjected t o lipouidase action a t a number of temperatures (Table I). The actual figures for the extinction coefficients obtained by Holman were not measured directly, but n-ere obtained by extrapolation of a VOL. 31, NO. 2, FEBRUARY 1959
299
0.57
a
$
0
5
0
IO
I5
,
20
I
25
I
30
35
- Isomerized
-Alkali
2 0.3
----Lipmidose
\
0.21
\\
- Isomerized
I
40
TIME (minutes)
Figure 2. Oxidation and conjugation of cis,cis-linoleic acid in the presence of a high concentration of trans-linoleic acid Table I. Effect of Temperature on Lipoxidase-Linoleate Extinction Coefficient
Temp. of
Repion, C 0 0.5 5
8
20 28 33 37
Molar Extinction Coefficient (234 &I,) Holman This (3) study 31,400 ... 2 i , Yo0 22,300 27,800 ... 2 i,600 ... 21,700 21,600 23,000 ...
combination of manometric and spectrophotometric data: the data from this report were obtained by direct reading of completely isomerized samples nith no assumption other than strict adherence to Beer’s law. Although the data in Table I indicate a slightly higher extinction coefficient a t the lower temperatures, the important finding is that over the range of temperatures where the quantitative enzymatic method will be used, temperature has no effect. Inhibition Studies. Holman (5) showed inhibition of lipoxidase by elaidolinolenic, 10,12-linoleic, oleic, and octanoic acids, and showed that these compounds act as competitive inhibitors. To test whether the method could be used on materials low in linoleic acid but high in inhibitors, a mixture of 1 part of linoleic acid and 50 parts of oleic acid was subjected to oxidation in the presence of lipoxidase. The amount of enzyme and the conditions of incubation described above were sufficient to bring about complete oxidation of the linoleic acid in the sample. As this is an extreme case, the conditions used should overcome any difficulties due to inhibitors. As a further check on this point, and assuming inhibition by trans isomers, a sample of linoleic acid was obtained which had been isomerized with sulfur dioxide catalyst to an average of 1.74 transbonds per molecule. The lipoxidase isomerization of the &,cis-linoleic acid
300
ANALYTICAL CHEMISTRY
0.lj
$
‘.
4
\
O i 220
--.-
I
1
240
260
I
_ _ _ _ _ _ , I
300
280 WAVELENGTH
Figure 3. Isomerization fatty acids
in this material was followed spectrophotometrically (Figure 2); the reaction was completed in 15 minutes. This finding also supports the evidence obtained in the presence of oleic acid, that the procedure given provides sufficient enzyme to overcome inhibition by competitive inhibitors. Extinction Coefficients and Structure. Because alkaline isomerization
of linoleate gives rise t o conjugated diene only, of linolenate to conjugated diene and triene, and of arachidonate to conjugated diene, triene, and tetraene (Figure 3), it was necessary to learn which conjugated species arose on the enzymatic isomerization of these same three compounds. Each gave rise to conjugated diene only (Figure 3). Siddiqi and Tappel (7) state that the molar extinction coefficient of urd bean lipoxidase-isomerizedlinoleic acid is considerably higher than that of the product from linolenate and arachidonate: 15,000, 7000, and 8100, respectively. They also say that Holman and Bergstrom (6) noticed the same correlation with soybean lipoxidase. In all these studies, the reactions had been followed manometrically and the ultraviolet absorption of the products was measured only after dilution with alcohol. The author believes Holman and Bergstrom to be correct in their esplanation for the differences in molar extinctions-the instability of the conjugated diene hydroperoxides arising from linolenate and arachidonate. When linoleate, linolenate, and arachidonate are subjected to the spectrophotometric analysis described, all yield single diene conjugations with the same molar extinction coefficients. That the
of
320
340
( mr)
polyunsaturated
extinctions of conjugated dienes arising from linoleate and linolenate are the same was demonstrated with a sample of unsaturated methyl esters from linseed oil. The alkaline isomerization and enzymatic analyses of this sample are given in Table I1,A. Because, in this instance, the standard for the enzymatic assay was linoleate, any real differences in extinction coefficients of conjugated linoleate and linolenate would have been evident, because this sample had more than four times as much linolenate as linoleate. Thus, the conjugated diene arising from lipoxidnse-catalyzed conjugation of linoleic acid has essentially the same extinction coefficient as the diene from linolenic acid. As arachidonate is tetraenoic, it is possible that two diene conjugations can arise from the lipoxidase-catalyzed conjugation of this acid. If so, one would expect arachidonate to yield about twice the extinction as an equal amount of linoleate after treatment of both with Iipoxidase. To test this, the fatty acids from hog (gilt) ovaries were isolated as a source of arachidonate. The arachidonate was concentrated by low temperature crystallization of the saturated methyl esters from acetone. Table 11, B, lists the analytical values obtained by both the enzymatic and alkaline techniques. There was excellent agreement between the two methods. Two thirds of the polyunsaturated fatty acid composition was due to arachidonate. If two dienes were formed from arachidonate, each molecule of arachidonate would be expected to yield about twice the extinction of an equal amount of linoleate. Thus,
the value for the enzymatically determined total polyunsaturated fatty acids, based on the extinction coefficient of linoleate, would have been about 5870, the sum of 6.5y0 linoleate; 0.3 linolenate; 42.2 arachidonate (instead of 21.1); and 8.8 pentaenoate (instead of 4.4). It can be safely assumed that only one conjugated diene is formed from each arachidonic acid molecule and that the extinction coefficient of conjugated arachidonic acid is essentially the same as that of conjugated linoleic and linolenic acids. Thus, the three most common polyunsaturated fatty acids all yield the same specific extinction coefficient when assayed by the lipoxidase method, and the lipoxidase method gives the total of polyunsaturated fatty acids containing the cis-methylene-interrupted diene structure. Extinction Coefficient Value. Samples of corn oil, cottonseed oil, soybean oil, and safflowerseed oil were analyzed for their polyunsaturated fatty acid content by the alkaline isomerization procedure of the American Oil Chemists’ Society ( I ) . Conjugation of these same samples of oils after saponification by the lipoxidase procedure permitted calculation of the lipoxidase specific extinction coefficient of the oils (Table 111). The values in the last column, the specific extinction coefficient of polyunsaturated fatty acids, were obtained arithmetically from the data in the other two columna. Precision. The value of the specific extinction coefficient of polyunsaturated fatty acids was determined on three separate occasions as illustrated in Table 111. The values were 77.8, 79.9, and 76.8, with a mean of 78.2. The coefficient of variation for these three determinations was 2.0. The coefficient of variation for 20 determinations of the specific extinction coefficient of purified linoleic acid was 13. SPECIFIC APPLICATIONS
Hydrogenated Oils. To test the method on hydrogenated oils, the series of mixtures of ethyl linoleate and hydrogenated vegetable oil listed in Table IT’ was assayed by the enzymatic method. The theoretical values were determined by summing the expected absorptions of t h e coniponents of the mixtures. The good agreement between the theoretical and observed values indicates that the presence of hydrogenated fats has no effect on the assay of the methylene-interrupted cis-polyunsaturated fatty acids in the mixture. Table V compares the data obtained by the alkaline isomerization technique and the lipoxidase method on hydrogenated fats. The agreement between the two methods is good, except for
Table II.
Ester A. Linseed
oil
Analyses of Unsaturated Methyl Esters Polyunsaturated Fatty Acid Content, Alkali Enzyisomer- matic Acid ization analysis Linoleic Linolenic Total
16.7
79.6 96.3
B. Hog ovary Linoleic 6.5 Linolenic 0.3 Arachidonic 2 1 . 1 Pentaenoic 4.4 Total 32.3
92.4
Table 111. Specific Extinction Coefficient of Polyunsaturated Fatty Acids Total PFA,a K 2 3 4 , b Alkali EnzyIGthod, matic Oil % Oil PFA !1.8 54.2 44.3 Cottonseed I 19.3 49.3 39.1 Cottonseed11 77.4 56.4 43.7 Corn I 80.7 54.3 43.8 Corn I1 Savhean T 57.6 46.8 81.2 So;bean
iI
Safflowerseed1 0
b
32.7
57.6 74 3
44 6 60.3
77.8 81.1 79 9
Mean Polyunsaturated fatty acids. Specific extinction coefficient at 234
mp
Recovery of Linoleate in Hydrogenated Vegetable Oil-Lipoxidase Method Ethyl Hydrogenated Polyunsaturated Fatty Acid Linoleate, -1 Veg. Oil, y Recovered, y Calcd., -( Recovery, 95
Table IV.
13.5 16.8
40.5 20.2
sample TII, which yielded a niiich lower value by the enzymatic method, probably because the high trans-polyunsaturated fatty acid content was partially assayed by the alkali isomerization method and not by the lipoxidase technique. This hydrogenated oil saniple (VII) had 25% trans-unsaturation calculated as elaidic acid. The alkali isomerization technique yielded results n ith hydrogenated fats I t o VI FT hich were very reliable as to cis-polyunsaturated fatty acid content. Direct Determination of Polyunsaturated Fatty Acids in Tissues. Where blood plasma and plant seeds nere analyzed directly for their polyunsaturated fatty acid contents, there n-ere very high, but readable, absorptions in the blanks. The absorptions of the blanks were too high to zero the Beckman DE spectrophotometer a t 234 nib. Of the alternatives open, a submaximal wave length was arbitrarily chosen for plasma, and a Cary recording spectrophotometer n as used for the plant seeds.
BLOODPLASMA.Saponify 0.1 ml. of rat blood plasma as described, but in a 25-ml. volumetric flask. Add 5 ml. of the 1.0M buffer and proceed as above, but measure the absorbance of the sample a t 245 instead of 234 nip and use a factor of 5460 instead of 3964. RIICROORGANISMS, Saponify 100 mg. of dried mycelium (Pencillium javanicum was used) a t room temperature for 24 hours, using 2 ml. of 1.5-1- alcoholic potasrium hydroxide. Add 20 ml. of
18.0 17.6
17.8 18.3
101.1 96.2
Table V.
Analyses of Hydrogenated Fats Polyunsaturated Fatty Acid, % Alkalj isomeriz- Enzymatic Sample ation method Hydrogenated fat I 32.4 30,4 I1 8.6 7.4 I11
10.6 29.5 19.0 11.4 10.3
9.8 27.4 17.4 10.1
6 0
the 1 . O M buffer, 6 ml. of 0.5s hydrochloric acid, and water to the 100-ml. mark. Filter through a dry filter paper, and dilute an aliquot of the filtrate tenfold with the 0.2M buffer. Proceed as with a free fatty acid solution. PLANT SEEDS. Weigh and slice five soft wheat kernels into five transverse sections each. Saponify in a 200-ml. volumetric flask with 4 ml. of 1.5N alcoholic potassium hydroxide by heating gently on a steam bath for % hours, then allowing to stand in the dark for 24 hours. Add 40 ml. of 1.0111 buffer and 6 ml. of 0.5X hydrochloric acid, dilute to volume nith water, and filter the final solution. For analysis of the polyunsaturated fatty acids, treat IO-ml. aliquots of the sample solution with 0.33-ml. aliquots of the enzymes, and measure the absorbance a t 234 mp in the Cary recording spectrophotometer. VOL. 31, NO. 2, FEBRUARY 1959
301
CONCLUSIONS
A spectrophotometric method for the total polyunsaturated fatty acids containing the cis-methylene-interrupted diene structure is based on a most specific type of reagent, the enzyme lipoxidase. Fats, oils, hydrogenated fats, fatty acids, esters, blood plasma, microorganisms, and plant seeds can be assayed directly for their total methylene-interrupted cis-polyunsaturated fatty acid content. As a routine analvtical procedure, tlic lipoxidase method should serve as
a valuable adjunct to the available analytical techniques. It is a micromethod, allowing for the accurate determination of as little as 57 of linoleic acid. LITERATURE CITED
(1) Am. Oil Chemists’ Soc., Tentative
Method Cd 7-48, rev. April 1956, “Official and Tentative Methods of the American Oil Chemists’ Society,” Chicago. (2) Brown, J. B., Frankel, J., J . d n a . Chena. Soc. 60, 54 (1938). ( 3 ) Holman, R. T., Arch. Biochena., 15, 402 (194i). (4) Holman, R. T., ”Methods of Bio-
chemical Analysis,” Vol. 11, p. 113, D. Click, ed., Interscience, New York, 1955. (5) Holman, R. T., Bergstrom, S., “The Enzvmes,” Vol. 11, Part I, p. 574, J. B. Sumrier and RI. Myrback, eds., Academic Press, S e w York, 1951. (6) Rlitchell, J. H., Jr., Kravbill, H. R., Zscheile, F. P., 1x0. EKG, CHEx., ANAL. ED. 15, 1 (1943). ( i )Siddiqi, 4.M., Tappel, A. L., J . A m . 0~1Chernzsts’ Soc. 34, 529 (1957). RECEIVEDfor review June 19, 1958. Accepted October 10, 1958. Federation of American Societies for Experimental Biologv, Chicago, Ill., April 1957, and Fourth International Conference on Biochemical Problems of Lipids, Oxford, England, Julj 1957.
Spot Tests for Aromatic Compounds Using 2,4,7-Tri nitrofluo renone H. T. GORDON and M. J. HURAUX Department of Entomology and Parasitology, University of California, Berkeley, Calif.
Many aromatic compounds form colored complexes when 10 y are spotted on filter paper and sprayed with 0.5% 2,4,7-trinitrofluorenone in benzene. Some of the complexes also show brilliant ultraviolet fluorescence. Qualitative information about the structure of unknown compounds can be derived very easily by observing the color and fluorescence of the trinitrofluorenone complex on paper, and by determining the solubility of the complex in iso-octane and ethyl alcohol. The trinitrofluorenone reaction is also useful for the detection of aromatic compound: on paper chromatograms.
compleses are formed 2,4,7-trinitrofluorenone u-hen (TKF)is mixed with various aromatic compounds, either as solutions in ethyl alcohol or benzene (6, 7) or as solids in the Kofler mixed fusion technique (3-5). The complexes usually are in a 1 to 1 molecular ratio, are readily crystallized, have a sharp melting point, and are intensely colored. The colors commonly range from yellow to red, the darker colors usually indicating either many fused aromatic rings (as in anthracene or perylene) or the presence of polar groups (as in catechol or aniline). Empirical rules for predicting whether an aromatic compound will react n ith TNF have been derised (4). The molecule must be relatively planar and not substituted with bulky groups (such as tert-butyl). Complex formaOLECULAR
302
ANALYTICAL CHEMISTRY
Table l.
Color, Fluorescence, and Heat Stability of Trinitrofluorenone Complexes on Filter Paper
Heatb Color” Fluorescencea 5 &lin,a t Sutural Kith T S F Xatural Kith TSF 110” C. Conipoimds with 3 or more fused rings 3-Aleth~~lcholanthrene G + B 0 Pyrene Ro (1,8)- or (4,5)-dimethylerienaphthalene R Chrysene
3,4-Benzoquinoline !,6-Benzoquinoline i,8-Benxoquinoline Phenanthrene 2-hlethylphenmthrene 2-Bcety lphenanthrene 9-Bromophenanthrene 4,shfet hylenephenanthrene Retene (isopropylphenanthrene) 9-Phenanthroic acid Phenant hrenequinone Thioanthrene Aceanthrene Anthracene Anthraquinone 1,8-Dihydroxyanthraquinone
Fluorant hene Fluorene Fluorenone 2-A4cetylaminofluorene
9-Methy1-2,3,i-trihydrosy-6-fluo-
rone Xanthene Xanthone Thioxanthone Acenaphthene Acenaphthenol Acenapht hylene Acenapht henequinone 9,10-Dihvdroacridine Thiodiphenylamine (phenothinzine) Rotenone
Reserpine Colchicine
++ ++ ++ +o ++ + 1-l+ Y ++ 1++ I++ 1++ 1+ YO +o ++I.1+o
++ RBI Gv -
++ YY ++ + y z!=
RO
++ 0 + y
=!=Y 10
+++ +o ++ 1-0 + Gy
++ G =tBrR
+1 YG 0
+
